Project Summary This proposal seeks competitive renewal of a multi-PI project (Fu and Yeo), which aims to develop innovative genomics approaches to elucidate regulatory pathways in the regulation of RNA metabolism. Built upon our accomplishments in the past funding cycles and aiming at addressing some emerging concepts in regulatory biology, we propose to pursue four specific aims: In Aim 1, we will develop a novel genomics technology to measure nascent RNA production at the level of single cells. Once fully developed, we will utilize this technology to advance the concept of transcription hubs in which genes may be expressed in a coordinated fashion and link promoters and enhancers within each hub to understand how long-distance regulatory DNA elements are looped into the proximity of gene promoters to control the amplitude and frequency of burst transcription. In Aim 2, we will continue the project initiated from the last funding cycle to characterize novel regulators for alternative polyadneylation (APA). From the proposed genome-wide screen, we have identified 5 gene networks involved in APA regulation, one of which correspond to a group of well-established splicing factors. We thus propose to pursue the mechanisms underlying the crosstalk between splicing and APA regulation. In Aim 3, we propose to develop new tethered function assays by constructing a library of ~1000 tethered-enabled RBPs to systematically characterize and identify RBPs involved in APA regulation. Given that mammalian genomes express several hundred zinc finger-containing proteins that have the capacity to bind RNA (based in our previous findings) and/or DNA, many of which were also identified from our genome-wide screens for splicing and APA regulators, we propose to devote Aim 4 to develop a large collection of genomically tagged cell lines to enable characterization of their transcriptome-wide binding to RNA and/or DNA and determine their functional impact on gene expression. This proposal combines both hypothesis-driven and discovery-driven research to address these outstanding problems in the regulation of gene expression.